Marker for pathologies comprising an auto-immune reaction and/or for inflammatory diseases

ABSTRACT

The present invention comprises a marker for inflammatory diseases and/or pathologies comprising an autoimmune reaction, which is the plasmatic membrane and/or a portion therof, in particular the plasmatic DNA. The present invention concerns also the method to obtain said marker and the diagnostic device which comprises said marker.

This is the U.S. national phase under 36 U.S.C. §371 of InternationalApplication PCT/BE95/00100, filed Oct. 30, 1995.

FIELD OF THE INVENTION

The present invention relates to a marker for pathologies comprising anauto-immune reaction and/or for inflammatory diseases, the antibodiesdirected against said marker and the antibodies directed against saidantibodies.

The present invention relates also to a diagnostic device whichcomprises said marker and/or said antibodies.

The present invention also relates to a method for obtaining the markerpresent in an animal cell.

BACKGROUND OF THE INVENTION

Numerous pathologies comprising an auto-immune reaction and inflammatorydiseases have an uncertain or unknown etiology and may have amultifactor origin.

Apparently, a disorder or a stimulation of the immune system of patientsconstitutes an important element of the progression of those diseases orpathologies.

Among the auto-immune diseases, systemic lupus erythematosus disease(SLE) is characterized by numerous biological and clinical signs.

However, SLE is a pathology with several clinical signs which may evolveand/or recover themselves each other (i.e. renal, cardiac, braindamages, . . . ). Therefore, as it exists non common and constant signsfor all the patients affected by SLE, it has been proposed that the"diagnostic" of this pathology is now obtained from simultaneouscharacterization of at least four clinical and/or biological signsrelated to SLE (Arthritis and Rheumatism, Vol. 25, No 11 (1982),Official Journal of the American Rheumatism Association Section of theArthritis Foundation).

The table 1 represents the sensitivity and the specificity of severalpreliminary criteria used for the diagnostic of systemic lupuserythematosus (serological and clinical tests).

                  TABLE 1                                                         ______________________________________                                        Comparison of sensitivity and specificity of elements                           in the 1982 and 1971 patient database                                                    Sensitivity.sup.1                                                                           Specificity.sup.2                                  Element      1982       1971   1982     1971                                  ______________________________________                                        Malar rash   101/177 (57)   (64) 156/162                                                                             (96) (98)                                Discoid rash 31/177 (18) (17) 161/162 (99) (99)                               Alopecia 99/177 (56) (43) 143/162 (88) (97)                                   Photosensitivity 76/176 (43) (37) 155/162 (96) (99)                           Oral ulcers 47/177 (27) (15) 155/162 (96) (99)                                Raynaud's 51/176 (29) (20) 132/162 (81) (99)                                  Arthritis 152/177 (86) (86) 60/162 (37) .sup. NA.sup.3                        Proteinuria 89/177 (50) (61) 148/157 (94) (82)                                Urinary casts 64/176 (36) (48) 152/157 (97) (89)                              Dementia 11/177 (6) NA 160/162 (99) NA                                        Seizures 21/177 (12) (13) 160/162 (99) (98)                                   Coma 8/177 (5) NA 162/162 (100) NA                                            Psychosis 22/176 (13) (19) 161/162 (99) (95)                                  Focal neurologic 21/177 (12) (11) 155/161 (96) (92)                           Pleurisy 92/177 (52) (60) 144/162 (89) (91)                                   Pericarditis 31/177 (18) (19) 155/162 (96) (97)                               Haemolytic anaemia 31/176 (18) (16) 160/161 (99) (98)                         Leucopenia 82/177 (46) (40) 144/161 (89) (94)                                 Thrombocytopenia 37/177 (21) (11) 160/161 (99) (98)                           LE cells 58/79 (73) (92) 46/48 (96) (98)                                      Sm antibody 34/108 (31) NA 59/62 (95) NA                                      Serologic test for syphilis 19/129 (15) (12) 80/80 (100) (99)                 Renal biopsy 57/69 (83) NA 10/10 (100) NA                                     Skin biopsy 47/69 (68) NA 13/16 (81) NA                                       Antinuclear antibody 174/175 (99) NA 68/139 (49) NA                           DNA antibody 113/168 (67) NA 84/91 (92) NA                                    CH50 84/120 (70) NA 33/47 (70) NA                                             C3 88/137 (64) NA 69/76 (91) NA                                               C4 65/102 (64) NA 33/51 (65) NA                                               C2 0/0  NA 0/0  NA                                                          ______________________________________                                         .sup.1 : Sensitivity was determined on the SLE population and is expresse     as the number of patients who were positive over the number of patients i     whom the test was determined. Number in parenthesis indicates percentage.     .sup.2 : Specificity was determined on the control population and is          expressed as the number of patients who were negative or normal over the      number of patients in whom the test was determined.                           .sup.3 : NA = data non available                                         

State of the Art

However, in order to improve the diagnostic of said pathology, effortshave been made to develop specific and reliable detection devices on thebasis of clinical biology analysis.

The presently used techniques are based on the detection in the serum ofanti-nuclear antibodies (ANA) directed against the autologous componentspresent in the cell nucleus. The presence of those anti-nuclearcomponents is associated to clinical manifestations of some auto-immunediseases.

The detection of those antibodies is generally effected previously toother tests for confirming the existence of an auto-immune disease.

The anti-nuclear auto-antibodies (ANA) can be divided into twosubgroups, the anti-DNA auto-antibodies and the anti-ENA auto-antibodies(extractable nuclear antigens).

A better characterization of those anti-nuclear auto-antibodies (ANA)would allow to obtain a more detailed diagnostic on the auto-immunedisease type.

Moreover, it has been shown that sera of SLE-affected patients contain amixture of antibodies respectively able to react not only with nuclearDNA, but also with RNA and nucleoproteins. Besides, some patientspresenting clinical signs of Systemic lupus erythematosus (SLE) have nosignificiant titers of anti-DNA antibodies.

Consequently, it has not been shown yet that the presence of suchantibodies in patients can be considered as a characteristic SLE marker.

In the European Patent EP-0252787 granted to Institut Pasteur andINSERM, a composition of isolated and purified cell surface polypeptidesand the application thereof for detecting SLE are described. Thesepurified polypeptides are characterised by a molecular weight less than60 KD (55 KD, 43 KD, 34 KD, 33 KD, 17 KD, 16 KD and 14 KD).

However, for the inflammatory diseases and the pathologies comprising anauto-immune reaction, particularly for SLE, it has not been yet possibleto provide a sufficiently specific antigenic structure to obtain areliable diagnostic (in specificity and in sensitivity) for thosepathologies and diseases.

Aims of the Invention

The present invention aims to provide a marker and a diagnostic deviceallowing to improve the diagnostic for inflammatory diseases and/orpathologies comprising an auto-immune reaction, particularly for SLEand/or Sjogren Syndrom.

A further aim is to provide a marker which improves the specificity andsensibility of SLE diagnostic, preferably an early diagnostic of SLE.

Another aim of the invention is to provide a method to obtain themarker, particularly a nucleic acid according to the invention.

A further aim of the invention is to provide an easy and reproduciblemethod to obtain said marker in high amounts.

SUMMARY OF THE INVENTION

The present invention relates to a marker for inflammatory diseasesand/or pathologies comprising an auto-immune reaction; said marker isthe plasmatic membrane and/or a portion thereof having a molecularweight higher than 60 KD, preferably higher than 100 KD, both obtainedfrom an animal cell containing molecular DNA (cmDNA).

Advantageously, said animal cell is a human blood cell, preferably alymphoblastoid B, such as a Wil 2 cell.

It is meant by "pathology comprising an auto-immune reaction"auto-immune diseases and/or infections leading to a disorder of theimmune system.

Auto-immunity is a an immunization state of a subject against its ownconstituents. Production by an organism of antibodies directed againstits own constituents has been observed in a number of pathologies suchas SLE affections, Gougerot-Sjogren syndrome (or Sjogren syndromepathology), rheumatoid polyarthritis, etc. It is obvious that theexpression "pathology comprising an auto-immune reaction" is in no waylimited to those diseases, but also includes other pathologies such assarcoidosis and osteopenia, spondylarthritis, scleroderma, multiplesclerosis, amyotrophic lateral sclerosis, hyperthyroidism, Addison'sdisease, auto-immune haemolytic anaemia, Crohn's disease, Goodpasture'ssyndrome, Graves' disease, Hashimoto's thyroiditis, idiopathic purpurahaemorrhagica, insulin-dependent diabetes, myasthenia, pemphigusvulgaris, pernicious anaemia, poststreptococcal glomerulonephritis,psoriasis, scleroderma and spontaneous sterility.

It is meant by "a portion of the plasmatic cell membrane", any fragmentobtained from said plasmatic cell membrane and having a molecular weighthigher than 60 KD, preferably higher than 100 KD.

Said portion (or epitope) may be any constituent of said plasmaticmembrane which can bind one or more antibodies present in the serum of apatient affected by an inflammatory disease and/or a pathologycomprising an auto-immune reaction.

Advantageously, said fragment is a nucleic acid, preferably a DNA, whichhas a molecular weight higher than 60 KD, preferably higher than 100 KD.

The nucleic acid present in said plasmatic cell membrane has beenpreviously described by Lerner et al. (Proc. Nat. Acad. Sci. U.S.A.,Vol. 68, No 6, pp. 1212-1216 (1971)).

According to Lerner et al., said specific DNA is not a viral, amycoplasmic, a mitochondrial or a nuclear nucleic acid.

The Inventors have discovered that the nucleic acid they have isolatedis from the plasmatic membrane not coming from apoptotic cells (theelectrophoresis obtained does not correspond to the electrophoresis of anuclear DNA).

Said DNA does not correspond to a mitochondrial DNA as the ORF₁ and ORF₄probes cannot hybridize to the DNA according to the invention.

As the viability of the Wil 2 cell is comprised between 95 and 98%, saidDNA cannot come from nuclear DNA of dead cell absorbed on the surface orthe Wil 2 cell.

According to Kuo et al. (Proc. Nat. Acad. Sci. U.S.A., Vol. 72, pp.5004-5006 (1975)), said specific DNA is probably of chromosomal origin(chromosome 9) and according to Meinke et al. (J. of Molecular Biology,Vol. 78, pp. 43-56 (1973)), said DNA has been also isolated from hepaticand other lymphoid cells.

Other characteristics of said DNA (synthesis, localisation,physicochemical properties, . . . ) were described by Hall et al.(Nature New Biology, Vol. 234, pp. 227-229 (1971)).

The Inventors have also demonstrated that the antibodies directedagainst said DNA are different from the anti-nuclear auto-antibodies(ANA). Bennet et al. (J. of Clin. Invest., Vol. 71, pp. 611-618 (1983),J. Rheumatol., Vol. 13, pp. 679-685 (1986) and Clin. Exp. Immunol., Vol.86, pp. 374-379 (1991)) have not been able to confirm that theanti-nuclear auto-antibodies (ANA) were able to bind the DNA obtainedfrom the membrane of human blood cells.

The results of Bennet et al. have been confirmed by Kubota et al.(Immunology Letters, Vol. 23, pp. 187-194 (1990)). Kubota et al. haveidentified upon the membrane, histone which is involved in thecross-reactivity of some anti-nuclear auto-antibodies (ANA) against themembrane of lymphocyte B cell line (Raji).

Therefore, said histone may bind exogenous DNA (Emlem et al., J.Immunol., Vol. 148, pp. 3042-3048 (1992)).

These results have been also confirmed by Muso and Jacob (Clin. Immunol.and Immunopathol., Vol. 42, pp. 370-374 (1987)). Muso and Jacob haveidentified the protein which is able to bind the histone DNA complex(Jacob et al., Proceeding of National Academy of Sciences USA, Vol. 86,pp. 4669-4673 (1989)).

Hereafter, the Inventors demonstrate that it is possible to obtainauto-antibodies purified from the serum of patients affected with SLE.Said auto-antibodies are obtained from a chromatographic process whereina cmDNA obtained from Wil 2 cells is fixed on a solid support in achromatographic process. Said auto-antibodies are specific for SLE andmay bind the DNA according to the invention in a ELISA test or inimmunofluorescence.

The Inventors have also demonstrated that these antibodies are differentfrom the anti-nuclear auto-antibodies (ANA).

The present invention is also related to the antibodies (polyclonal ormonoclonal) or a portion thereof directed against said marker and to theantibodies (polyclonal or monoclonal) or a portion thereof directedagainst said antibodies anti-marker.

The term "a portion thereof" means a fragment of said antibodies (suchas the Fab'₂ portion) which is able to bind specifically the marker orthe antibodies according to the invention.

The present invention is also related to a diagnostic device, such as akit or a chromatographic column, which comprises the marker and/or theantibodies or a portion thereof according to the invention.

Preferably, the diagnostic device also comprises the reactants fordetection and/or dosage of antibodies or nucleotides sequences through amethod selected from the group consisting in the in situ hybridization,hybridization or recognition by marked antibodies such as ELISA (EnzymeLinked Immunosorbent Assay) or RIA (Radio Immunoassay) method on filter,on a solid support, in solution, in "sandwich", on gel, Dot blothybridization, Northern blot hybridization, Southern blot hybridization,by an isotonic or non-isotopic labelling (such as immunofluorescence orbiotynilation), by a technique of cold probes, by genetic amplification,particularly PCR or LCR, by a double immunodiffusion, by acounter-immunoelectrophoresis, by haemagglutination, by enzymeconjugation and/or a mixture thereof.

Another aspect of the present invention relates to a method forobtaining the marker according to the invention wherein the plasmaticmembrane is isolated from animal cells, preferably from human bloodcells, particularly from lymphoblastoids B, such as the Wil 2 cells.

Advantageously, the membrane is isolated from the animal cell throughits fixation upon a solid support by the addition of an organic solvent.

Preferably, said method comprises also the further steps, wherein theisolated membrane is treated by an enzyme selected from the groupconsisting of RNase, DNase, pronase (or peptidase) lipase, glycosidaseand/or a mixture thereof, and wherein the marker obtained is recovered.

The present invention is also related to a process for the detection invitro or antibodies related to inflammatory diseases and/or pathologiescomprising an auto-immune reaction, preferably systemic lupuserythematosus and/or Sjogren, in a biological fluid containing saidantibodies, the process comprising the following steps:

the biological fluid, preferably a serum, is put in contact with themarker according the invention or to a marker obtained by the methodaccording to the invention and/or to an antibody or portion thereofdirecting against the antibodies (directed against the marker accordingto the invention), in order to obtain an immunological binding in vitrobetween the antibody present in the biological fluid and said marker orsaid antibody,

and detection in vitro of the binding obtained.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 and 2 show electrophoresis gels of the human membranousdouble-stranded DNA possibly preincubated with specific haemoglobulins(IgG) of SLE having undergone various treatments,

The invention will be further described in the following non limitingexamples.

EXAMPLES

Materials

1. Cell Lines

The cell lines of human lymphoblastoids B (Wil 2 NS) obtained from ICNFlow Laboratories (ECACC No 90112121) are maintained in RPMI 1640 addedwith 10% foetal calf serum (heat inactivated and tested for the absenceof mycoplasmas), L Glutamine and 40 μg/ml Gentamycine, humid oven at 37°C. and 5% CO₂. Cells are removed in the growing logarithmic phasecorresponding to a cell density of 1-1,2 10⁶ cells/ml and showing aTrypan Blue exclusion of more than 95%.

2. Sera

The sera are obtained from patients affected by several inflammatorydiseases or pathologies having an auto-immune reaction and from normalindividuals. The sera are obtained from centrifuged coagulated blood andheld at -20° C. until use.

Methods

1. Indirect Immunofluorescence on Cells

1.1. Immunofluorescence on Methanol Fixed Cells

Wil 2 cells are washed twice with Hank's solution (Gibco BRL) andresuspended at 0.5×10⁶ /ml and subsequently spotted on glass slidesdivided into 20 μl wells.

After drying at 37° C. in an oven, the cells are fixed for 3 min. inmethanol and washed once in PBS (Phosphate Buffered Saline, 10 mM pH7.4). The cells are then incubated for 30 min. at room temperature inthe presence of goat IgG (Fc fraction, Organon Technica) at 6 μg/well in20 μl PBS, then washed in PBS. They are then incubated for 25 min. atroom temperature in the presence of different dilutions of patients orcontrol sera at 20 μl/well and washed again in PBS. In some experiments,as noted, an IgG fraction is used instead of whole sera.

The slides are further incubated for 30 min. in the presence of 20 μl offluorescein-conjugated goat anti-human IgG (Kallestadt) diluted 1/50 inPBS.

A final wash of the slides is performed with PBS alone, followed by PBScontaining Evans Blue as counterstain.

Finally, slides are mounted in glycerin/PBS (2:1), pH 9, and visualizedby means of a UV immersion microscope (Leitz Orthoplan),magnification=10×40).

Scoring of the patterns observed were as follow:

a negative results ("0") corresponds to the absence of any membranefluorescence,

a cell membrane punctate pattern is arbitrarily referred as "1", and

a cell membrane peripheral continuous ring is scored as "2".

1.2. Immunofluorescence on Non-fixed Cells

The reactions are performed on cells suspensions at 4° C. to reducereceptor turnover and internalization. Briefly, cells (1×10⁶) in 1 ml ofculture medium are placed in plastic (5 ml) tubes and washed with PBScontaining 10 mM EDTA and 0.01% Na azide (PBS EA) by centrifugation(500×g, 10 min.).

The pellet is resuspended with 600 μg of goat IgG in 900 μl of the samebuffer and incubated for 30 min. at 4° C.

Subsequently, cells are washed twice in the same buffer as above. Seraor purified IgG for either patients or controls are incubated atdifferent dilutions with the cells for 30 min. at 4° C. A new washingcycle is performed, followed by incubation with fluorescein-conjugatedgoat anti-human IgG (Kallestadt), diluted 1/50 in 200 μl of PBS EA.

Four washes are performed in the same conditions as described above, andthe cell pellet is resuspended in PBS EA containing Evans Blue ascounterstain. 10 μl of this cell suspension are placed onto a glassslide, covered and visualized by UV microscopy.

1.3. DNase, RNase and Pronase Treatment of Cells

After methanol fixation of the slides, DNase RQ1®, RNase free (Promega)is incubated (20 μg in 20 μl of PBS) with the cells for 60 min. at roomtemperature. The RNase treatment (RNase I®, Promega; 2 μg in 20 μl ofPBS) is also performed for 60 min. but at 37° C.

After two washings of the slides in PBS, the subsequent steps of theprocedure are performed as described above.

The pronase treatment (10 μl from sigma) was performed for 1 hour at 37°C. The subsequent steps of the procedure are performed as describedabove.

2. ELISA Upon Isolated Membrane from Wil 2 Cells

The Wil 2 cells membranes are obtained according to the method describedby Bennet et al. (J. Clin. Invest., Vol. 71, pp. 611-618 (1983)).

3.1. DNA (cmDNA) Isolated from Wil 2 Cells Membrane

1×109 cells Wil 2 are pooled together into two tubes from said pelletsre-suspended in a PES buffer (10 mM salt phosphate buffer, pH 7.2) andwashed with 50 ml of the same buffer (500×g, 10 minutes). Two otherwashings are then carried out with 50 ml per tube of RBS buffer (1 mlHCl 1M, 0.2 ml NaCl 5M, 0.2 ml MgCl₂ 1M extended up to one liter withdistilled water). Each pellet obtained is re-suspended in 10 ml RBS and30 μl NP₄ O are added to each tube. After an incubation of 10 minutes atroom temperature, the tubes are centrifuged at 2000×g during 4 minutes.The supernatants are submitted to centrifugation with same conditionsand after a transfer into new tubes (the following elements being addedto each supernatant: 0.32 ml EDTA 0,5M, 3.2 ml NaCl 5M, 1 ml 10% SDS),the volume in each tube is adjusted to 16.7 ml.

After an overnight incubation at 4° C., the supernatants of acentrifugation (20000×g, 30 minutes, 4° C.) are extracted twice withphenol and extracted with chloroform: isoamyl alcohol (24:1). A 3 mincentrifugation at 2000×g is carried out between each step to separatethe phases. Two volumes of absolute ethanol are added per volume of theaqueous phase obtained. After an overnight incubation at 4° C. and a 30min centrifugation at 20000×g, the pellet is re-suspended in 0.2 ml TEbuffer (Tris 10 mM, EDTA 1 mM, pH 8).

An electrophoresis on 1% agarose of the resulting product shows an DNAband of ±17000 base pairs and two slighter RNA bands (corresponding to±2000 and 1000 pairs of bases).

The use of those cell lineages of lymphoblastoids B makes possible toobtain easily a human-specific "antigenic" structure.

3.2. Enzymatic Treatment of Said Purified DNA (cmDNA)

However, as that structure can be formed with DNA associated with RNAand proteins, an one hour digestion is carried out at 37° C. with RNaseI® provided by Promega.

To 100 μl of the preparation, 50 U/5 μl RNase and 25 ml buffer 10 mMTris HCl, 5 mM EDTA, 200 mM sodium acetate, pH 7.5, are added. Thereaction is stopped by addition of 10 μl NaCl 5M.

A new phenol extraction is then carried out to remove the enzyme and thepreparation is passed through a column of 1 ml Sephadex G50 ("spuncolumn") so as to remove the oligonucleotides resulting from the RNAdigestion.

Another digestion with pronase is carried out to confirm the absence ofprotein traces associated with the membrane DNA.

To 100 μl of the product digested by the RNase 5 μg/10 μl pronase areadded during 1 hour at 37° C.

Two extractions with phenol followed by an extraction with chloroform:isoamyl alcohol (24:4) are then carried out and the aqueous phase issent through Sephadex G50. An electrophoresis on 1% agarose is carriedout to confirm the so obtained membrane DNA size.

By comparing with the size of the DNA fragments digested by HindIII, themembrane DNA size is estimated to ±17000 pairs of bases.

The membrane DNA is then considered as being pure, that meansadvantageously free of contaminants such as RNA or proteins which caninterfere in the recognition phenomenon with antibodies directed againstRNA.

3.3. ELISA Upon the Isolated DNA (cmDNA) Treated or not by Enzymes

The Wells of a microplate "IMMUNOPLATE MAXISORB®" (NUNC) are coated with100 μl poly-L-Lysine (10 μg/ml) during 2 hours at 37° C.

Then, 100 μl purified membrane DNA, digested by RNase and pronase (15mg/ml), are incubated in wells for 3 hours at 37° C.

A saturation of the Wells is carried out through incubation of asolution of 10% horse serum supplied by Pharmacia in a buffer PBS (150μl/wells, 2 hours, 37° C.).

The serum of lupus-affected or control patients (blood donors) is thenincubated in the wells in series dilutions (100 μl of the dilutions from1/20 to 1/1280 in buffer PBS) overnight at room temperature.

100 μl of rabbit immunoglobulines (IgG) directed against the humanimmunoglobulines (IgG) and conjugated with peroxidase (DAKO), 1/250diluted in buffer PBS, are added to each well during 2 hours at 37° C.

The last step is a calorimetric reaction formed with the "substrate"solution (30 μl H₂ O₂) (Perhydrol®, Merck) and 17.5 mg orthophenylenediamine in 20 ml buffer PBS).

A washing of the cupules is carried out between each step by buffer PBS.

Results

1. Purified DNA (cmDNA) from Wil 2 Membranes

The FIGS. 1 and 2 show pictures of electrophoresis gels of the resultingpreparations (possibly incubated with lupus-specific immunoglobulines atdifferent dilutions) and treated with different enzymes, particularlyphospholipase, pronase, RNase and DNase. The resulting products arecompared to DNA size markers.

The FIG. 2 shows that the raw preparation contains a few proteinsremoved by pronase (column 4), but it is possible to obtain an RNA-freepure product (column 5).

Column 6 clearly shows that the resulting active product is constitutedby DNA.

Other production methods can be used to obtain a contaminant-free humanmembranous nucleic acid, particularly DNA.

It is particularly possible to obtain special derivatives of saidnucleic acid, such as fragments or epitopes of said nucleic acid.

A purification process can include an affinity chromatography usingspecific antibodies directed against all or part of that nucleic acidbound to a solid phase.

Said nucleic acid or fragments thereof can be also purified byelectrophoresis on a convenient gel.

The antibodies directed against said nucleic acids and the antibodiesdirected against said antibodies can be obtained by immunization ofanimals and produced in high amounts through selection of particularhybridomes.

2. Effect of the Preincubation of Immunoglobulines (IgG) fromLupus-affected or Healthy Patients with the Purified Membrane DNA on itsMigration on Agarose Gel

As shown by the picture of the FIG. 1, the preincubation of membrane DNAwith the various dilutions of purified immunoglobulines (IgG) from serumof a lupus-affected patient, shows a migration delay proportional to theadded IgG quantity.

The digestion of this mixture by pronase cancels or reduces stronglythat delay. In opposition, the same experience made with the samequantities of purified immunoglobulines (IgG) from a serum of a blooddonor shows no effect on the electrophoretic migration of membrane DNA.

3. Effect of the Predigestion of Cells with RNase or DNase on theBinding of the IgGs from Lupus-affected Patients to Plasma Membranes ofCells Wil 2

Table 2 represents the percentage of fluorescent cells giving eachimages ("0"=no fluorescence, "1"=point membranous fluorescence,"2"=continuous membranous fluorescence). The given values are thearethmetic average results of 6 experiments.

Cell treatment with DNase gives for sera of patients 1, 2, 6 a reductionof the cell percentage giving an image "2", whereas, for the sera ofpatients 3, 4, 5, there is shown either no enzyme effect or even anincrease of the cell percentage "2".

That could be due to the fact that the fluorescence image groupstogether various recognized specificities on the membrane of cells Will2, which specificities include membrane DNA.

Treatment with RNase causes per se an increase of the cell percentagegiving image "2", suggesting that RNA, maybe associated with membraneDNA, could partly mask the specific sites for those antibodies.

4. Fluorescence Obtained on Membranes of Cells Wil 2 After Binding ofSerum, IgG Fraction or Specific IgG Fraction of Membrane DNA

Table 3 demonstrates that the serum fraction responsible for thefluorescent image is due to immunoglobulines (IgG). Moreover, themembrane DNA specific IgGs allow sometimes to obtain a fluorescentimage. It is to be pointed out at that time that the membrane DNAspecific IgGs are used at lower concentration than the total IgGs.

                  TABLE 2                                                         ______________________________________                                        Images observed by fluorescence further to serum                                incubation of lupus-affected patients on cells Wil 2                                    "2"          "1"    "0"                                           ______________________________________                                        Control     0            0      100                                             + DNase 0 0 100                                                               + RNase 0 96 4                                                                Patient SLE 1 86 21 3                                                         + DNase 3 92 5                                                                + RNase 94 3 3                                                                Patient SLE 2 59 23 18                                                        + DNase 27 43 30                                                              + RNase 65 18 17                                                              Patient SLE 3 11 20 79                                                        + DNase 17 28 55                                                              + RNase 53 35 30                                                              Patient SLE 4 19 25 56                                                        + DNase 18 12 70                                                              + RNase 31 33 36                                                              Patient SLE 78 9 13                                                           + DNase 79 4 17                                                               + RNase 79 9 12                                                               Patient SLE 6 21 24 55                                                        + DNase 6 22 72                                                               + RNase 55 28 17                                                              Patient SLE 7 89 2 9                                                          + DNase 1 15 24                                                               + RNase 94 1 5                                                              ______________________________________                                         Notes:                                                                        image "0" = no fluorescence                                                   image "1" = point membranous fluorescence                                     image "2" = continuous membranous fluorescence                           

The first line of results corresponds to the average (on 6 experiments)of the cell number showing each fluorescent image. The second line(DNase) and the third line (RNase) show the percentage of those cellsafter a predigestion either with DNase or RNase.

                  TABLE 3                                                         ______________________________________                                        Immunofluorescence images due to binding to                                     cells                                                                         Wil 2 of serum, total IgGs or specific marker IgGs                              Serum, IgG or                                                               anti-marker IgG Bound Cells Unbound Cells                                   ______________________________________                                        Control serum  0         0                                                      IgG 0 0                                                                       Serum SLE 1 2 2                                                               IgG 2 2                                                                       IgG-marker 1 1 + 2                                                            Serum SLE 2 2 2                                                               IgG 2 2                                                                       IgG-marker 1 1 + 2                                                            Serum SLE 3 2 2                                                               IgG 2 1                                                                       IgG-marler 0 0                                                              ______________________________________                                    

This table shows the results of three different experiments.

                  TABLE 4                                                         ______________________________________                                        Results in fluorescence obtained from sera of                                   patients with SLE upon three different epitopes obtained                      from the membrane of Wil 2 cells (in percentages).                            Image "2"        Image "1" Image "0"                                        ______________________________________                                        Epitope 1: Fixed cells                                                          86               21        3                                                  59 23 18                                                                      11 20 79                                                                      19 25 56                                                                      78 9 13                                                                       21 24 55                                                                      89 2 9                                                                        Average: 51 Average: 17 Average: 32                                         Epitope 2: Fixed cells treated by DNase                                         3                92        5                                                  27 43 30                                                                      17 28 55                                                                      18 12 70                                                                      79 4 17                                                                       6 22 72                                                                       1 15 24                                                                       Average: 23 Average: 34 Average: 42                                         Epitope 3: Fixed cells treated by RNase                                         94               3         3                                                  65 18 17                                                                      53 35 30                                                                      31 33 36                                                                      79 9 12                                                                       55 28 17                                                                      94 1 5                                                                        Average: 66 Average: 18 Average: 16                                         ______________________________________                                         Image "2" for a seric dilution of 1/40                                   

These results indicate that the enzymatic treatment of the membranemodifies its ability to bind the antibodies.

For the diagnostic of SLE, the immunofluorescence is characterized by aspecificity of 100% and a sensibility of 43%, and for the detection ofSjogren syndrome, the sensibility is 3%.

5. Detection of Membrane Anti-DNA Antibodies by ELISA Technique inDifferent Auto-immune Diseases

The physiological fluid (serum) of the following patients has beentested with the ELISA diagnostic device according to the invention:

68 lupus

34 Sjogren syndrome

15 ankylosing spondylarthritis

17 osteopenia

44 healthy donors

5.1. Characteristics of the ELISA Device

Specificity:

The ELISA is specific, as, if auto-antibodies (IgGs) are purified byaffinity and are added to a serum of a healthy donor, the opticaldensity in ELISA changes from 0.241 to 0.898.

Sensitivity:

up to 16 AU/ml cc, which corresponds to a serum dilution of 1/3200 of apositive serum.

Reproducibility:

intra-assay:

CV<5% calculated on optical densities of the curve,

CV<6% calculated on arbitrary units (patient sera),

inter-assay:

CV<10% calculated on arbitrary units (patient sera)

CV=10% on slopes of 4 standard curves obtained in 4 different assays atdifferent days

Positivity threshold:

69 AU/ml (average +2 sd of 44 donors)

5.2. Dosage of the Marker with ELISA Diagnostic Kit

By definition, the healthy donors do not have any anti-membrane IgG.Osteopenia-affected patients are negative, lupus-affected patients havehigh levels and Sjogren syndrome-affected patients have well higherlevels than lupus.

A particularly high diagnostic is also observed withsarcoidosis-affected patients.

                  TABLE 5                                                         ______________________________________                                                                       Sarcoid-                                                                            Osteo-                                                                              Healthy                              Total CmDNA SLE Sjogren SPA osis penia patients                             ______________________________________                                        cmDNA    36/68   15/17   7/9   10/13 3/17  0/12                                 without RNA 33/68 25/34 7/9 10/13 4/17 0/37                                   and protein                                                                   cmDNA 35/68 17/17 8/9 11/13 4/7  0/12                                         without RNA                                                                   cmDNA  0/68  0/17 0/9  0/13 0/7  0/12                                         treated                                                                       with DNase                                                                  ______________________________________                                    

Therefore, it seems that the antigenicity of the marker is modifiedaccording to the enzymatic treatment of said marker.

In addition, the optical density measured with ELISA is modified whenthe marker (membrane) is modified by an enzymatic treatment.

                  TABLE 6                                                         ______________________________________                                        Marker             Optical density                                            ______________________________________                                        Non-treated membrane                                                                             0.660                                                        Membrane treated with DNase 0.508                                             Membrane treated with RNase 0.673                                           ______________________________________                                    

The following table 7 describes the dosage of membrane anti-DNA withELISA diagnostic kit.

                  TABLE 7                                                         ______________________________________                                        Osteopenia  SLE     SPA    Sjogren syndrome                                                                        Sarcoidosis                              ______________________________________                                        Av.   54        102     136  176       178                                      AU/ml                                                                         sd  2  3  3  3  3                                                           ______________________________________                                    

5.3. Correlation between the auto-antibodies calculated by ELISA andimmunofluorescence on Wil 2 cells

The levels by ELISA are correlated with the increase of the % of image"1" and "2" and a reduction of the negative image percentage "0".

    ______________________________________                                                 Images obtained by immunofluorescence                                ELISA      0           1         2                                            ______________________________________                                        IgG < 69 AU                                                                              123         15        2                                              IgG > 69 AU 87 22 10                                                        ______________________________________                                    

Chi Squared 9.47 p=0.0088

Taking pathologies into account:

    ______________________________________                                        ELISA      SLE      Sjogren syndrome                                                                          Osteopenia                                    ______________________________________                                        IgG < 69 AU                                                                              35       9           13                                              IgG > 69 AU 33 25 4                                                         ______________________________________                                    

Chi Squared 49 p=0.00001

When ELISA levels and immunofluorescence images are related fordifferent pathologies:

    ______________________________________                                                Healthy                                                                 patients SLE  Sjogren Osteopenia                                            ______________________________________                                        IF         0       0     1  2     0  1   2    0                                 ELISA < 69 AU 37 22 11 2  9 0 0 13                                            ELISA > 69 AU  0 14 12 7 23 1 1  4                                          ______________________________________                                    

Chi Squared 55 p<0.00001

That means that in lupus the higher levels with ELISA are correlatedwith an image "2" whereas in Sjogren syndrome the higher levels withELISA are correlated with an image "0".

5.4. Correlation Between Anti-marker (cmDNA) Auto-antibodies Levels withELISA and Single- or Double-stranded Anti-nuclear DNA Antibodies (ANA)Levels with ELISA

262 sera were measured with 3≠ELISA and classified according to thelevel of their antibodies.

Distributions are different, as both assays do not measure the sameantibody specificities.

    ______________________________________                                                        Membrane anti-cmDNA IgG                                         with ELISA                                                                                  <69 AU/ml                                                                             >69 AU/ml                                             ______________________________________                                        ANA IgG (DNA                                                                            < 40 AU/ml  140       102                                             ds) with > 40 AU/ml 3 17                                                      ELISA                                                                         ANA IgG (DNA < 40 AU/ml 122 83                                                ss) with > 40 AU/ml 21 36                                                     ELISA                                                                       ______________________________________                                    

ANA IgG (DNA ds) with ELISA Squared chi 12.0 p<0.0005

ANA IgG (DNA ss) with ELISA Squared chi 8.35 p<0.004

Many sera of high anti-cmDNA IgG level are negative in ANA (DNA ds orss).

6. Longitudinal Study of Lupus-affected Patients/Anti-cmDNA IgG withELISA and by Immunofluorescence Compared to Anti-ANA (DNA ds and ss)with ELISA

    ______________________________________                                                Time in months                                                                0    6           8       10                                           ______________________________________                                        IF 1/40   "2"    "2"         "2"   "2"                                          DNA ds 338 225 398 262                                                        DNA ss 347 262 >400   386                                                     cmDNA 362  72 170  78                                                       ______________________________________                                    

    ______________________________________                                               Time in months                                                                0    1        2     4       5    6                                     ______________________________________                                        IF 1/40  "0"    "0"      "1" "0"     "0"  "1"                                   DNA ds 5281 178 61  26  46 57                                                 DNA ss  145 145 56  49  61 77                                                 cmDNA 1651 228 55 186 203 55                                                ______________________________________                                    

DESCRIPTION OF FIGURES

FIG. 1

1. lambda HindIII markers

2. phospholipase digested mDNA

3. pronase digested mDNA

4. IgG lupus 1/20 preincubated mDNA

5. IgG lupus 1/20 preincubated mDNA, and then pronase digestion

6. IgG lupus 1/5 preincubated mDNA

7. IgG lupus 1/5 preincubated mDNA, and then pronase digestion

8. Ecori markers

9. DNase digested pure mDNA

10. RNase and pronase digested pure mDNA

FIG. 2

1. Void

2. HindIII

3. raw preparation

4. pronase digested raw preparation

5. RNase digested raw preparation

6. DNase digested raw preparation

We claim:
 1. A diagnostic kit for inflammatory disease or pathologycomprising an auto-immune reaction, comprising:an antibody specific formembranous DNA or an antigen-binding portion of an antibody specific formembranous DNA; and reactants for detecting said antibody or portionspecific for membranous DNA.
 2. The diagnostic kit according to claim 1,wherein said reactants for detecting said anti-membranous DNA antibodiesfunctions in a method selected from the group consisting of in situhybridization, hybridization, and recognition by marked specificantibodies, said method being conducted on filter, on solid support, insolution, or on gel, by using at least one technique selected from thegroup consisting of a sandwich method, Dot blot hybridization, isotopicor non-isotopic labelling, cold probe techniques, doubleimmunodiffusion, counter-immunoelectrophoresis, and hemagglutination. 3.The diagnostic kit according to claim 1, wherein the animal cell is ahuman blood cell.
 4. The diagnostic kit according to claim 3, whereinthe human blood cell is a lymphoblastoid B cell.
 5. The diagnostic kitaccording to claim 4, wherein said human blood cell is a Wil 2 cell. 6.A process for in vitro detection of an antibody related to inflammatorydisease or pathology comprising an auto-immune reaction, comprising thefollowing steps:(a) placing a biological fluid, in which said antibodymay be present, in contact with at least either:a marker obtained froman animal cell, said marker being a plasmatic membrane containing amembranous DNA or being a portion of said membrane containing orconsisting of a membranous DNA, said marker having a molecular weighthigher than 60 kD or an anti-marker antibody or antigen-binding portionthereof specific for said membranous DNA in order to obtain either abiological binding in vitro between said antibody present in thebiological fluid and said marker, or a competitive immunological bindingin vitro between said antibody present in the biological fluid and saidanti-marker antibody or said antigen-binding portion thereof specificfor said membranous DNA; and (b) detecting in vitro the bindingobtained.
 7. A process according to claim 6, wherein the pathologycomprising an auto-immune reaction is systemic lupus erythematosus orSjogren syndrome.
 8. A process according to claim 7, wherein saidbiological fluid is a serum of a patient having inflammatory disease orpathology comprising an auto-immune reaction.
 9. The process accordingto claim 6, wherein the animal cell is a human lymphoblastoid B cell.10. The process according to claim 9, wherein the human lymphoblastoid Bcell is a Wil 2 cell.
 11. The process according to claim 6, wherein saidmarker is membranous DNA.
 12. The process according to claim 6, whereinthe biological fluid is a serum.
 13. A reagent set for identifyinginflammatory disease or pathology comprising an auto-immune reaction,comprising:a biological fluid from a patient suspected of having saidinflammatory disease or pathology, said biological sample containinganti-membranous DNA antibodies when said inflammatory disease orpathology is present; a marker obtained from an animal cell, said markerbeing a portion of a plasmatic membrane comprising or consisting of amembranous DNA, said marker having a molecular weight higher than 60 kD;and reactants for detecting said anti-membranous DNA antibodies bound tosaid marker.
 14. The diagnostic kit according to claim 13, wherein saidmarker consists of said membranous DNA.
 15. The diagnostic kit accordingto claim 13, wherein said portion is a lysed cell membrane.